Conformational Change and Intermediates in the Unfolding of α1-Antichymotrypsin

Mary C Pearce,Harvey Rubin,Stephen P Bottomley

doi:10.1074/jbc.m004310200

Mary C Pearce, Harvey Rubin + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m004310200

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Abstract

Serpins are the prototypical members of the conformational disease family, a group of proteins that undergoes a change in shape that subsequently leads to tissue deposition. One specific example is alpha(1)-antichymotrypsin (ACT), which undergoes misfolding and aggregation that has been implicated in emphysema and Alzheimer's disease. In this study we have used guanidine hydrochloride (GdnHCl)-induced denaturation to investigate the conformational changes involved in the folding and unfolding of ACT. When the reaction was followed by circular dichroism spectroscopy, one stable intermediate was observed in 1.5 m GdnHCl. The same experiment monitored by fluorescence revealed a second intermediate formed in 2.5 m GdnHCl. Both these intermediates bound the hydrophobic dye ANS. These data suggest a four-state model for ACT folding N <--> I(1) <--> I(2) <--> U. I(1) and I(2) both have a similar loss of secondary structure (20%) compared with the native state. In I(2), however, there is a significant loss of tertiary interactions as revealed by changes in fluorescence emission maximum and intensity. Kinetic analysis of the unfolding reaction indicated that the native state is unstable with a fast rate of unfolding in water of 0.4 s(-1). The implications of these data for both ACT function and associated diseases are discussed.

Highlights

Conformational disorders, as defined by Carrell and colleagues [1, 2], arise when a protein undergoes a change in shape that leads to self-association, tissue deposition, and disease
When the reaction was followed by circular dichroism spectroscopy, one stable intermediate was observed in 1.5 M guanidine hydrochloride (GdnHCl)
In this study we have used a combination of spectroscopic approaches, including intrinsic tryptophan fluorescence and circular dichroism spectroscopy, to monitor the structural changes that occur within ACT as it unfolds and folds under equilibrium conditions

Summary

Introduction

Conformational disorders, as defined by Carrell and colleagues [1, 2], arise when a protein undergoes a change in shape that leads to self-association, tissue deposition, and disease. RCL sheet insertion is possible in the absence of proteolytic cleavage, two examples of which are the adoption of the latent conformation [13], and the formation of serpin polymers [14]. The folding pathways of small proteins have received immense attention, primarily due to the ease with which data can be obtained and interpreted [16]. These studies, for a small number of proteins, have revealed at high resolution how a linear polypeptide chain folds to its unique three-dimensional structure. Kinetic characterization demonstrates that the serpins are unstable and designed for dramatic conformational change

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